A large number of human and other mammalian proteins, including, for example, human growth hormone, human protein C and clotting Factor VII, have been produced in host cells by transfecting these cells with DNA encoding these proteins and growing the recombinant cells under conditions favorable for the expression of the protein. Grinnell et al. describe the expression of recombinant human protein C (HPC) by human kidney cells in Biotechnology, 5:1189-1192 (1987). The proteins are secreted by the cells into the cell culture medium, and must be separated from the culture medium and the other components, such as cell waste products, cell debris and proteins or other material, which also has collected in the medium. In addition, the biological activity of the protein must be preserved, so the recovery conditions must be mild enough to preserve the biological activity of the protein, but, at the same time, thorough enough to effectively separate the protein from contaminants in the medium. Purity is often an important consideration, especially for pharmaceutical applications.
Recovery of proteins in biologically active form from cell culture medium presents a number of problems. For example, the desired protein must be separated from other closely related proteins in the cell culture medium, such as homologous, biologically inactive proteins, which may be associated with the protein. The recovery process should yield the biologically active form of the protein with a high level of purity.
Jones et al. describe a method for recovering refractile proteins (non-exported proteins which form insoluble protein granules within the host cell) from the cytoplasm of a host cell in U.S. Pat. No. 4,512,922. Related patents describing denaturing-refolding protein recovery systems include U.S. Pat. Nos. 4,599,197; 4,518,526; and 4,511,503.
Raush and Meng in U.S. Pat. No. 4,677,196 describe recovery of heterogenous proteins from a host cell, which are also in the form of refractile bodies.
Hung et al. in U.S. Pat. No. 4,734,362 describe a process for recovering recombinant refractile proteins from a host cell, involving denaturing the protein, and subsequent renaturing to yield the desired product.
The recovery and purification of human coagulation Factor VII is described by Brose and Majerus in The Journal of Bioloqical Chemistry, 255:1242-1247 (1980). They purified Factor VII from human plasma with a yield of about 30% using a process which involved first absorbing the proteins to barium citrate, and separating by chromatography.
Vitamin K-dependent proteins are a class of proteins involved in maintaining hemostasis. The dependency on vitamin K occurs during the biosynthesis of the proteins. Human protein C (HPC) is a vitamin K-dependent plasma glycoprotein that plays a key role in maintaining hemostasis. C. T. Esmon, Science, 235:1348-1352 (1987).
The binding of calcium ions (Ca.sup.2+) to HPC causes a conformational change in HPC that can be measured by fluoresence emission spectroscopy. Johnson et al , J. Biol. Chem., 258:5554-5560 (1983). The conformational change results in a change in surface charge distribution as measured by a difference in the migration pattern of the protein in an electrical field, such as agarose gel electrophoresis. Stenflo, J., J. Biol. Chem., 251:355-363 (1976).